Method of deriving progenitor cell line

ABSTRACT

We disclose a method comprising: (a) providing an embryonic stem (ES) cell; and (b) establishing a progenitor cell line from the embryonic stem cell; in which the progenitor cell line is selected based on its ability to self-renew. Preferably, the method selects against somatic cells based on their inability to self-renew. Preferably, the progenitor cell line is derived or established in the absence of co-culture, preferably in the absence of feeder cells, which preferably selects against embryonic stem cells. Optionally, the method comprises (d) deriving a differentiated cell from the progenitor cell line.

CROSS REFERENCE

This application is a continuation application of pending U.S. patentapplication Ser. No. 12/065,549, filed on Mar. 3, 2008 and entitled“Method of Deriving Progenitor Cell Line,” which is a national phaseentry of PCT Application No. SG2006/000233, filed on Aug. 15, 2006,which claims priority to U.S. Provisional Patent Application Ser. No.60/713,992, filed on Sep. 2, 2005.

The foregoing application, and each document cited or referenced in eachof the present and foregoing applications, including during theprosecution of each of the foregoing application (“application andarticle cited documents”), and any manufacturer's instructions orcatalogues for any products cited or mentioned in each of the foregoingapplication and articles and in any of the application and article citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or reference indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text or in anydocument hereby incorporated into this text, are hereby incorporatedherein by reference. Documents incorporated by reference into this textor any teachings therein may be used in the practice of this invention.Documents incorporated by reference into this text are not admitted tobe prior art.

FIELD

The present invention relates to the fields of development, cellbiology, molecular biology and genetics. More particularly, theinvention relates to a method of deriving progenitor cells fromembryonic stem cells.

BACKGROUND

Stem cells, unlike differentiated cells have the capacity to divide andeither self-renew or differentiate into phenotypically and functionallydifferent daughter cells (Keller, Genes Dev. 2005; 19:1129-1155; Wobusand Boheler, Physiol Rev. 2005; 85:635-678; Wiles, Methods inEnzymology. 1993; 225:900-918; Choi et al, Methods Mol Med. 2005;105:359-368).

The pluripotency of mouse embryonic stem cells (ESCs) and their abilityto differentiate into cells from all three germ layers makes embryonicstem cells an ideal source of cells for regenerative therapy for manydiseases and tissue injuries (Keller, Genes Dev. 2005; 19:1129-1155;Wobus and Boheler, Physiol Rev. 2005; 85:635-678). However, this veryproperty of embryonic stem cells also poses a unique challenge, i.e.generating the appropriate cell types for the treatment of a specificdiseased or injured tissue in sufficient quantity and homogeneity toensure therapeutic efficacy, and inhibiting the generation of other celltypes that may have a deleterious effect on the tissue repair andregeneration. At present, protocols that either enhance differentiationof embryonic stem cells towards specific lineages and/or enrich forspecific tissue cell types are too inefficient and generally yieldheterogeneous cell populations that might be tumorigenic (Keller, GenesDev. 2005; 19:1129-1155; Wobus and Boheler, Physiol Rev. 2005;85:635-678).

This invention seeks to solve this and other problems with methods inthe art.

SUMMARY

According to a 1^(st) aspect of the present invention, we provide amethod comprising: (a) providing an embryonic stem (ES) cell; and (b)establishing a progenitor cell line from the embryonic stem cell; inwhich the progenitor cell line is selected based on its ability toself-renew.

Preferably, the method selects against somatic cells based on theirinability to self-renew.

In a preferred embodiment, the progenitor cell line is derived orestablished in the absence of co-culture, preferably in the absence offeeder cells. Preferably, the absence of co-culture selects againstembryonic stem cells.

In preferred embodiments, the progenitor cell line is establishedwithout transformation. Preferably, the progenitor cell line isestablished by exposing embryonic stem cells or their descendants toconditions which promote self-renewal of putative progenitor cells.Preferably, the self-renewal-promoting conditions discourage thepropagation of embryonic stem cells.

Preferably, the self-renewal-promoting conditions comprise growth inrich media. More preferably, the self-renewal-promoting conditionscomprise growing cells in the absence of LIF.

Preferably, the self-renewal-promoting conditions comprise serialpassages. Preferably, the self-renewal promoting conditions comprise atleast 12 serial passages.

In preferred embodiments, the progenitor cell line has reduced potentialcompared to the embryonic stem cell. Preferably, the progenitor cellline is lineage restricted, preferably non-pluripotent. Preferably, theprogenitor cell line is non-tumorigenic.

Preferably, the step of deriving the progenitor cell line comprises astep of exposing the embryonic stem cell to conditions that enhancedifferentiation to a specific lineage. Preferably, the differentiationenhancing-conditions comprises generating an embryoid body from theembryonic stem cell. Preferably, the cells are removed fromdifferentiation enhancing-conditions after pluripotency is lost.

Preferably, the removing of the cells from lineage restriction-promotingconditions comprises disaggregating an embryoid body. Preferably, themethod comprises disaggregating embryoid bodies which have been grownfrom between about 3 to 6 days.

In preferred embodiments, the progenitor cell line displays reducedexpression of or does not substantially express either or both of OCT4and alkaline phosphatase activity.

Preferably, the progenitor cell line displays reduced expression of apluripotency marker compared to an embryonic stem cell from which it isderived, the pluripotency marker preferably selected from the groupconsisting of: Nanog,BMP4, FGF5, Oct4, Sox-2 and Utf1.

In preferred embodiments, the progenitor cell lines display one or moreof the following characteristics: (a) are maintainable in cell culturefor greater than 40 generations; (b) have a substantially stablekaryotype or chromosome number when maintained in cell culture for atleast 10 generations; (c) have a substantially stable gene expressionpattern from generation to generation.

Preferably, the progenitor cell line does not substantially induceformation of teratoma when transplanted to a recipient animal,preferably an immune compromised recipient animal, preferably after 3weeks, more preferably after 2 to 9 months.

Preferably, the embryonic stem cell or progenitor cell line is amammalian, preferably mouse or human, embryonic stem cell or progenitorcell line.

Preferably, the progenitor cell line comprises an endothelial progenitorcell line, preferably a E-RoSH cell line. Alternatively, or in addition,the progenitor cell line may comprise a mesenchymal progenitor cellline, preferably a huES9.E1 cell line.

In some embodiments, the method further comprises the step of (d)deriving a differentiated cell from the progenitor cell line.

Preferably, the progenitor cell line is propagated for at least 5generations prior to differentiation.

There is provided, according to a 2^(nd) aspect of the presentinvention, a method according to the 1^(st) aspect of the invention forgenerating a differentiated cell from an embryonic stem (ES) cell.

Preferably, the differentiated cell is an endothelial cell or amesenchymal cell. More preferably, the differentiated cell is anadipocyte or an osteocyte.

We provide, according to a 3^(rd) aspect of the present invention, amethod according to the 1^(st) or 2^(nd) aspect of the invention forup-regulating expression of mesenchymal or endothelial markers of acell.

As a 4^(th) aspect of the present invention, there is provided a methodaccording to the 1^(st) or 2^(nd) aspect of the invention fordown-regulating expression of stem cell or pluripotency markers of acell.

We provide, according to a 5^(th) aspect of the present invention, amethod of identifying an agent capable of promoting or retardingself-renewal or differentiation of a stem cell, the method comprisingperforming a method according to any preceding aspect of the inventionin the presence of a candidate molecule, and determining an effectthereon.

The present invention, in a 6^(th) aspect, provides a method accordingto any preceding aspect of the invention for the production of aprogenitor cell line or a differentiated cell for the treatment of, orthe preparation of a pharmaceutical composition for the treatment of,any one of the following: a disease treatable by regenerative therapy,cardiac failure, bone marrow disease, skin disease, burns, degenerativedisease such as diabetes, Alzheimer's disease, Parkinson's disease andcancer.

In a 7^(th) aspect of the present invention, there is provided aprogenitor cell line produced by a method according to any precedingaspect of the invention.

According to an 8^(th) aspect of the present invention, we provide adifferentiated cell produced by a method according to any precedingaspect of the invention.

We provide, according to a 9^(th) aspect of the invention, a method ofgenerating a differentiated cell from an embryonic stem (ES) cell, themethod comprising: (a) deriving a progenitor cell line from theembryonic stem cell; (b) propagating the progenitor cell line; and (c)deriving a differentiated cell from the progenitor cell line.

There is provided, in accordance with a 10^(th) aspect of the presentinvention, a method comprising: (a) providing an embryonic stem (ES)cell; (b) deriving a progenitor cell from the embryonic stem cell; and(c) establishing a progenitor cell line from the progenitor cell, inwhich progenitor cells are selected based on their ability toself-renew.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Derivation of E-RoSH cell lines.

FIG. 1A. Embryonic stem cells are plated singly on methycellulose basedmedia to form embryoid bodies (EBs). At day 3-6, embryoid bodies areharvested, dissociated by collagenase and cultured as a monolayer ongelatinized feeder plate. RoSH-like colonies with adherentfibroblast-like cells and ring-like structures are selected andpropagated on gelatinized plates to generate E-RoSH 1, 2, 3 . . . . Eachof the cultures are then plated at a low density of 10-100 cells per 10cm plate and single RoSH like colonies are picked to establishedsublines, E-RoSH 2.1, 2.2, 2.3 . . . etc.

FIG. 1B. A putative RoSH-like colony consisting of adherent shortfibroblast-like cells with characteristic ring-like cells (inset)expanding over time.

FIG. 1C. Morphological similarity between E-RoSH2.1 and RoSH2 cells insub-confluent cultures;

FIG. 1D. Alkaline phosphatase staining of E-RoSH2.1 and its parental E14embryonic stem cells;

FIG. 1E. Average chromosome number from 20 metaphase nuclei in E-RoSH2.1and 3.2 lines at passage 3 and 13;

FIG. 2. Relative gene expression analysis by quantitative RT-PCRanalysis. The expression level is normalized against that of embryonicstem cells and expressed as a logarithmic function.

FIG. 2A. Gene expression profile of E-RoSH2.1 cells at three differentpassages;

FIG. 2B. Comparative gene expression profiles in E-RoSH2.1, E-RoSH3.2and RoSH2 cells;

FIG. 2C and FIG. 2D Relative expression of genes associated withpluripotency and endothelial potential in the parental E14 embryonicstem cells and E-RoSH2.1 cells as measured by quantitative RT-PCRanalysis.

FIG. 3. Characterization of E-RoSH cells.

FIG. 3A. In vitro differentiation of RoSH2.1 cells on matrigel coatedplate. In two weeks, RoSH2.1 cells differentiate to form a network oftubular structures that covered the surface of the entire tissue dish;

FIG. 3B. E-RoSH derived tubular structures have patent lumens andendocytosed acetylated LDL. The structures are labeled with CFDA, acytoplasmic green fluorescent dye (Molecular Probe, Eugene, Oreg.) andpropidium iodide, and viewed by confocal microscopy (left panel). Thetubular structures are incubated with acetylated red fluoresecentdiI-labelled LDL (Molecular Probe, Eugene, Oreg.) for 24 hours andcounterstained with SYTOX Green™, a green fluorescent nuclear dye(Molecular Probe, Eugene, Oreg.) before analysis by confocal microscopy;

FIG. 3C. Immunoreactivity for vWF on paraffin-embedded sections ofE-RoSH2.1 derived tubular structures are using HRP-based detectionsystem. Brown precipitates indicate positive staining. The nuclei arestained with Mayer's hematoxylin.

FIG. 3D. Gene expression during endothelial differentiation of E-RoSH2.1cells as measured by quantitative RT-PCR analysis. Relative geneexpression is normalized against that at time 0 and expressed as alogarithmic function.

FIG. 3E. Suspension cultures of embryonic stem cells and E-RoSH at day 7

FIG. 3F. Quantitative RT-PCR profiling of gene expression by embryonicstem cells and E-RoSH2.1 cells when cultured in suspension cultures for0, 2, 3 and 7 days. Relative gene expression is normalized against thatof embryonic stem cells at time 0 and expressed as a logarithmicfunction.

FIG. 3G. In vivo differentiation. 1×10⁵ E-RoSH cells labeled with Qdot®nanocrystals (655 nm emission) are injected into a embryonic stemcell-derived teratoma that is induced in SCID mice. Three days later,the mice are euthanized and the tumors are removed. The tumors are fixedin 4% paraformaldehyde and cryosectioned at 20 μm thickness. Thesections are assayed for pecam-1 immunoreactivity using rat anit-pecam1followed by FITC-conjugated rabbit anti-rat antibody, and counterstainedwith DAPI. The sections are viewed by light microscopy and then confocalmicroscopy.

FIG. 4. Characterization of HuES9.E1 cells.

FIG. 4A. HuES9 colony grown on mitotically inactive MEFs surrounded byproliferating fibrobastic stromal cells (arrow).

FIG. 4B. A representative confluent culture of HuES9.E1 MSC-like cellsand BM-derived MSCs.

FIG. 4C. HuES9, a human embryonic stem cell line, E14, a mouse embryonicstem cell line, mouse embryonic fibroblast (MEF) and HuES9.E1mesenchymal stem cell (MSC)-like cells are stained for the presence ofalkaline phosphatase activity.

FIG. 4D. HuES9 and HuRS9.E1 MSC-like cells are tested for the expressionof Pou5f1 by quantitative RT-PCR analysis using TaqMan® primers. Pou5f1transcript level in HuES9 human embryonic stem cell normalized to one.

FIG. 4E. Genomic PCR analysis for the presence of human-specific Alurepeat sequence and mouse-specific c-mos repeat sequences in HuES9.E1MSC-like cells.

FIG. 4F. Karyotype analysis of HuES9.E1 at passage 4 and passage 8.

FIG. 4G. Profile of surface antigens by FACS analysis. HuES9.E1 cellsare tested for immunoreactivity against CD29, CD44, CD105, CD166, CD34and CD45.

FIG. 4H. Differentiation of HuES9.E1 into adipocytes and osteocytes.Confluent HuES9.E1 cells are cultured in standard culture media forinducing adiogenesis or osteogenesis. After 12 days, cells that areinduced to undergo adiogenesis are stained for oil droplets by oil redand analyzed for the expression of PPARγ by quantitative RT-PCR (toppanel) while those that are induced to undergo osteogenesis are stainedfor calcium deposits by von Kossa staining and analyzed for theexpression of bone-specific alkaline phosphatase, ALP by quantitativeRT-PCR (bottom panel).

DETAILED DESCRIPTION

We demonstrate that it is possible to derive progenitor cell lines fromembryonic stem cells (ES), based on the ability of progenitor cells toself-renew. Unlike terminally differentiating cells, putative progenitorcells with self-renewing properties can be selected and propagatedwithout transformation.

Our methods therefore generally involve deriving progenitor cell linesof limited potential from embryonic stem cells by culturing thepluripotent cells in vitro. This enables the expansion of a progenitorcell with a highly restricted differentiation potential that, upondifferentiation, will generate a highly enriched population of aspecific cell type with reduced or abolished tumorigenic potential.

We therefore use this property of self-renewal of progenitor cells asthe underlying principle of self-selection for generating lineagerestricted progenitor cell lines from embryonic stem cells.

Absence of Co-Culture

In preferred embodiments, however, the method further includes cultureof cells in conditions that promote growth of progenitor cells, andoptionally retard or prevent growth or propagation of embryonic stemcells.

Thus, in highly preferred embodiments, our methods involve culturingputative progenitor cells in the absence of co-culture, as a monolayeror in the absence of feeder cells. The term “co-culture” refers to amixture of two or more different kinds of cells that are grown together,for example, stromal feeder cells. According to preferred embodiments ofthe methods described here, the embryonic stem cells are cultured in theabsence of feeder cells to establish a progenitor cell line.

Biasing Differentiation

In preferred embodiments, the method for generating embryonic stemcell-derived progenitor cell lines of specific lineages preferablyfurther comprises a first step of biasing differentiation of embryonicstem cells towards a specific desired lineage or lineage of interest.Our methods may also comprise a second step of encouraging self-renewalof putative progenitor cells and discouraging the propagation ofembryonic stem cells.

The first step may comprise promoting the growth or propagation of aspecific lineage of interest. Different progenitor cell lines ofspecific lineages of interest may be made by exposing the cells toconditions that promote the differentiation of those lineages ofinterest. For example, the embryonic stem cells may be exposed to growthfactors or small molecules such as ligands that promote or enabledifferentiation.

Thus, the methods described here for establishing embryonic stemcell-derived cell lines of specific lineages preferably include a stepof enhancing differentiation of embryonic stem cells towards thatspecific lineage. Preferably, the differentiation-enhancing step iscarried out for a predetermined period of time. Thus, preferably, theembryonic stem cells or their descendants are transiently exposed todifferentiation-enhancing environment.

The choice of the method of enhancing or biasing differentiation willdepend on the specific cell lineage of interest for which it is desiredto produce progenitor cells. The person skilled in the art will be awareof the various methods which may be used for different cells.

Endodermal Progenitor Cells

Where it is desired to bias differentiation of embryonic stem cellstowards endodermal types of tissues, for example, embryoid bodies may beformed and disaggregated (see later). The disaggregated embryoid bodiesmay be exposed to growth factors or drugs or combinations thereof thatinduce endodermal differentiation. Examples of such growth factors anddrugs include activin A, FGF4, dexamethasone and retinoic acid.

Hematopoietic and Endothelial Progenitor Cells

On the other hand, where it is desired to bias differentiation ofembryonic stem cells towards hematopoietic or endothelial lineages, thedisaggregated embryoid bodies may be exposed to growth factors or drugsor combinations thereof that induce hematopoietic or endothelialdifferentiation. Examples of such growth factors and drugs includeGM-CSF, G-CSF, SCF, PDGF, IL-3, erythropoietin, thrombopoeittin, TNFαand rapamycin.

Cardiac Mesoderm and Skeletal Myoblast Progenitor Cells

On the other hand, where it is desired to bias differentiation ofembryonic stem cells towards cardiac mesoderm or skeletal myoblastlineages, the disaggregated embryoid bodies may be exposed to growthfactors or drugs or combinations thereof that induce cardiac mesoderm orskeletal myoblast differentiation. Examples of such growth factors anddrugs include dexamethasone, inhibitors of PPARγ and testosterone or itsanalogs.

The second step may comprise plating the differentiating cells in a richmedia. In such embodiments, continued propagation will selectivelyenrich for progenitor cells which can then be cloned.

Formation of Embryoid Bodies

In some embodiments, the differentiation-enhancing step comprisesformation of embryoid bodies from embryonic stem cells. Embryoid bodies,and methods for making them, are known in the art. The term “embryoidbody” refers to spheroid colonies seen in culture produced by the growthof embryonic stem cells in suspension. Embryoid bodies are of mixed celltypes, and the distribution and timing of the appearance of specificcell types corresponds to that observed within the embryo. Preferably,the embryoid bodies are generated by plating out embryonic stem cellsonto semi-solid media, preferably methylcellulose media as described inLim et al, Blood. 1997; 90:1291-1299. Preferably, the embryoid bodiesare between 3 to 6 days old.

In such embodiments, the embryoid body is disaggregated, i.e.,separating the component cells from each other, e.g., by collagenase ortrypsin treatment, in order to remove the cells from lineagerestriction-promoting conditions.

The method in preferred embodiments comprises a step of choosing aputative progenitor cell for the desired specific lineage. The choosingmay be conducted based on morphology of the cell, or by expression ormarkers, etc. Gene expression profiling or antigen profiling may also beused to choose specific progenitor cells which are of a desired lineage.The chosen putative progenitor cell for the desired specific lineage maythen be cultured, or further choosing steps conducted thereon.

In preferred embodiments, the differentiation-enhancing step is followedby exposing differentiating cells to conditions which encourageself-renewal of putative progenitor cells and discourage the propagationof embryonic stem cells. Such conditions may preferably comprise culturein the absence of co-culture or feeder cells (see above).

Rich Media

Alternatively, or in addition, such conditions comprise plating in richmedia. The term “rich media” as used in this document is intended torefer to media which is nutrient rich. Preferably, such media comprisesessential nutrients required for growth of the relevant cell.Preferably, the rich media contain serum. More preferably, it comprisessubstantially all the nutrients required for such growth. Mostpreferably, the rich medium supports, promotes and encourages growth ofthe relevant cells. in highly preferred embodiments, the relevant cellis a progenitor cell or a putative progenitor cell of interest. Anexample of a rich medium is DMEM with 4500 mg/l D-glucose, supplementedwith 20% fetal calf serum, non essential amino acids, L-glutamine andβ-mercaptoethanol.

In preferred embodiments, such rich media does not comprise additionalgrowth regulators or hormones that allow, promote or encourage growth ofembryonic stem cells, such as Leukemia Inhibitory Factor (LIF).

According to such embodiments, continued propagation will selectivelyenrich for progenitor cells which can then be cloned.

Long-Term Maintenance in Culture

Preferably, the methods described here involve culturing the embryonicstem cells or their descendants for more than one generation.Preferably, the cells are cultured for more than 5, more than 10, morethan 15, more than 20, more than 25, more than 50, more than 40, morethan 45, more than 50, more than 100, more than 200, more than 500 ormore than 800 generations. In particular, the cell lines may bemaintained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 200, 500 or more generations.

Cells in culture will generally continue growing until confluence, whencontact inhibition causes cessation of cell division and growth. Suchcells may then be dissociated from the substrate or flask, and “split”or passaged, by dilution into tissue culture medium and replating. Theprogenitor cells may therefore be passaged, or split during culture;preferably they are split at a ratio of 1:2 or more, preferably 1:3,more preferably 1:4, 1:5 or more. The term “passage” designates theprocess consisting in taking an aliquot of a confluent culture of a cellline, in inoculating into fresh medium, and in culturing the line untilconfluence or saturation is obtained.

The progenitor cells derived according to the methods described here mayhowever be maintained for a large number of generations, based on theircapacity to self-renew. On the other hand, it has been established that“normal” (i.e., untransformed somatic) cells derived directly from anorganism are not immortal. In other words, such somatic cells have alimited life span in culture (they are mortal). They will not continuegrowing indefinitely, but will ultimately lose the ability toproliferate or divide after a certain number of generations. On reachinga “crisis phase” such cells die after about 50 generations. Thus, suchsomatic cells may only be passaged a limited number of times.

Importantly, the progenitor cells are able to maintain self-renewalwithout the requirement for transformation. Thus, for example, knowntransformation treatments such as fusion with immortalised cells such astumour cells or tumour cell lines, viral infection of a cell line withtranforming viruses such as SV40, EBV, HBV or HTLV-1, transfection withspecially adapted vectors, such as the SV40 vector comprising a sequenceof the large T antigen (R. D. Berry et al., Br. J. Cancer, 57, 287-289,1988), telomerase (Bodnar-A-G. et. al., Science (1998) 279: p. 349-52)or a vector comprising DNA sequences of the human papillomavirus (U.S.Pat. No. 5,376,542), introduction of a dominant oncogene, or by mutationare therefore not required in the methods described here for makingprogenitor cell lines.

According to preferred embodiments of the methods described here,progenitor cells may be propagated without transformation for more than50 generations. In preferred embodiments, the progenitor cells may bepropagated indefinitely and without transformation as progenitor celllines. The progenitor cells and progenitor cell lines are preferablylineage restricted compared to their parental embryonic stem cells. Inparticular, they are not capable of giving rise to all three germlayers. In highly preferred embodiments, the progenitor cell lines arepreferably non-pluripotent.

Characteristics of Progenitor Cells

In preferred embodiments, the progenitor cells and cell lines (or thedifferentiated cells derived from them) do not display one or morecharacteristics of embryonic stem cells. Preferred such characteristicsinclude expression of the OCT4 gene and alkaline phosphatase activity.Preferably, the progenitor cell line exhibits reduced expression of oneor more characteristic markers of pluripotency. Such pluripotencymarkers are described in further detail below, but include Nanog, BMP4,FGF5, Oct4, Sox-2 and Utf1.

Progenitor cells made by the methods described here are preferablynon-tumorigenic. Preferably, the progenitor cells when implanted into animmune compromised or immunodeficient host animal do not result intumours, compared to implantation of parental embryonic stem cells whichresults in tumour formation. Preferably, the immune compromised orimmunodeficient host animal is a SCID mouse or a Rag1 −/− mouse.Preferably, the progenitor cells do not form tumours after prolongedperiods of implantation, preferably greater than 2 weeks, morepreferably greater than 2 months, most preferably greater than 9 months.Detailed protocols for tumourigenicity testing are set out in theExamples.

Progenitor cells made by the methods described here are also preferablydisplay one or more of the following characteristics. They have asubstantially stable karyotype as assessed by chromosome number,preferably when maintained in cell culture for at least 10 generations.They also preferably display a substantially stable gene expressionpattern from generation to generation. By this we mean that theexpression levels one or more, preferably substantially all, of a chosenset of genes does not vary significantly between a progenitor cell inone generation and a progenitor cell in the next generation.

Preferably, the set of genes comprises one or more, a subset, or all of,the following: cerberus (GenBank Accession nos: NM_(—)009887, AF031896,AF035579), FABP (GenBank Accession nos: NM_(—)007980, M65034, AY523818,AY523819), Foxa2 (GenBank Accession nos: NM_(—)010446, X74937, L10409),Gata-1 (GenBank Accession nos: NM_(—)008089, X15763, BC052653), Gata-4(GenBanlc Accession nos: NM_(—)008092, AF179424, U85046, M98339,AB075549), Hesx1 (GenBank Accession nos: NM_(—)010420, X80040, U40720,AK082831), HNF4a (GenBank Accession nos: NM_(—)008261, D29015,BC039220), c-kit (GenBank Accession nos: NM_(—)021099, Y00864, AY536430,BC075716, AK047010, BC026713, BC052457, AK046795), PDGFRα (NM_(—)011058,M57683, M84607, BC053036), Oct4 (GenBank Accession nos: NM_(—)013633,X52437, M34381, BC068268), Runx1 (GenBank Accession nos: NM_(—)009821,D26532, BC069929, AK051758), Sox17 (GenBank Accession nos: NM_(—)011441,D49474, L29085, AK004781), Sox2 (GenBank Accession nos: NM_(—)011443,U31967, AB108673), Brachyury (NM_(—)009309, X51683), TDGF1 (GenBankAccession nos: NM_(—)011562, M87321) and Tie-2 (GenBank Accession nos:NM_(—)013690, X67553, X71426, D13738, BC050824).

The methods described here enable the production of progenitor cells andprogenitor cell lines as well as differentiated cells, which compriseclonal descendants of progenitor cells. The term “clonal descendant” ofa cell refers to descendants of the cells which have not undergonesubstantially any transforming treatment or genetic alteration. Suchclonal descendants have not undergone substantial genomic changes aresubstantially genetically identical to the parent cell, or an ancestor,preferably, the embryonic stem cell (save with reduced potency). Theterm “progenitor cell” should also preferably be taken to include celllines derived from progenitor cells, i.e., progenitor cell lines, andvice versa.

Regulators of Self-Renewal and Differentiation

Our methods may also be used to identify putative regulators ofself-renewal or differentiation. The methods involve conducting themethods described for production of progenitor cell lines ordifferentiated cells in the presence and absence of a candidatemolecule, and identifying if the presence of the molecule has any effecton the process. For example, a molecule which accelerates the productionof progenitor cells or differentiated cells may be used as a positiveregulator of differentiation (or alternatively as an inhibitor ofself-renewal). Conversely, a molecule which retards the process can beconsidered an inhibitor of differentiation or a promoter ofself-renewal.

In preferred embodiments, we also provide a cell, preferably aprogenitor, of a selected lineage, obtainable according to the method.Hitherto, preparations of progenitors were too impure for certainty asto whether any chosen cell was a progenitor cell. With culture accordingto the invention that can give rise to substantially 100% purepreparations of progenitors, isolation of a single progenitor isachieved.

We further provide in preferred embodiments a composition comprising aplurality of cells, wherein a majority of the cells are progenitor cellsof a selected lineage. Preferably, at least 60% of the cells areprogenitor cells of the selected lineage. More preferably, at least 60%of the cells are progenitor cells. In addition, the invention providesan isolated progenitor cell. The term cell line preferably refers tocells that can be maintained and grown in culture and display animmortal or indefinite life span.

The methods described here may be combined with decreasing the activityof mTOR to promote differentiation, as described in U.S. 60/609,216,herein incorporated by reference.

Progenitor Cells and Stem Cells

The methods described here are capable of producing progenitor cells,and cell lines thereof.

When embryonic stem cells differentiate, they generally recapitulate thecomplexity of early mammalian development where embryonic stem cellstransit through a series of lineage restriction to generate progenitorcells of decreasing lineage potential before finally generatingterminally differentiated cells representing all three germ layers(Wiles, Methods in Enzymology. 1993; 225:900-918). This is exemplifiedby the process of hematopoiesis, where increasingly lineage-restrictedhematopoietic progenitors appearing in a sequential manner similar tothat found within the mouse embryo, can be identified within embryoidbodies (Choi et al, Methods Mol Med. 2005; 105:359-368).

Typically, stem cells generate an intermediate cell type or types beforethey achieve their fully differentiated state, referred to as aprecursor or progenitor cell. Progenitor or precursor cells in foetal oradult tissues are partly differentiated cells that divide and give riseto differentiated cells. Such cells are usually regarded as “committed”to differentiating along a particular cellular development pathway,Progenitor cells are therefore sometimes referred to as “committed stemcells”.

Our methods are capable of producing of progenitor cells and cell linesof various types.

For example, we disclose a method of making peripheral blood progenitorcells (PBPC), neuronal progenitor cells, haematopoeitic progenitorcells, myeloid progenitor cells, epithelial progenitor cells, bonemarrow stromal cells, skeletal muscle progenitor cells, pancreatic isletprogenitor cells, mesenchymal progenitor cells, cardiac mesodermal stemcells, lung epithelial progenitor cells, liver progenitors, andendodermal progenitor cells.

Progenitor cells made according to the methods described here can beused for a variety of commercially important research, diagnostic, andtherapeutic purposes. These uses are generally well known in the art,but will be described briefly here.

For example, stem cells may be used to generate progenitor cellpopulations for regenerative therapy. Progenitor cells may be made by exvivo expansion or directly administered into a patient. They may also beused for the re-population of damaged tissue following trauma.

Thus, hematopoietic progenitor cells may be used for bone marrowreplacement, while cardiac progenitor cells may be used for cardiacfailure patients. Skin progenitor cells may be employed for growing skingrafts for patients and endothelial progenitor cells for endothelizationof artificial prosthetics such as stents or artificial hearts.

Embryonic stem cells and their tissue stem cell derivatives may be usedas sources of progenitor cells for the treatment of degenerativediseases such as diabetes, Alzheimer's disease, Parkinson's disease,etc. Stem cells, for example may be used as sources of progenitors forNK or dendritic cells for immunotherapy for cancer, which progenitorsmay be made by the methods and compositions described here.

It will be evident that the methods and compositions described hereenable the production of progenitor cells, which may of course be madeto differentiate using methods known in the art. Thus, any uses ofdifferentiated cells will equally attach to those progenitor cells forwhich they are sources.

Progenitor cells produced by the methods and compositions described heremay be used for, or for the preparation of a pharmaceutical compositionfor, the treatment of a disease. Such disease may comprise a diseasetreatable by regenerative therapy, including cardiac failure, bonemarrow disease, skin disease, burns, degenerative disease such asdiabetes, Alzheimer's disease, Parkinson's disease, etc and cancer.

We therefore describe a method of treatment of a disease comprising: (a)providing an embryonic stem (ES) cell; (b) establishing a progenitorcell line from the embryonic stem cell in which the progenitor cell lineis selected based on its ability to self-renew; (d) optionally derivinga differentiated cell from the progenitor cell line; and (e)administering the progenitor cell line or the differentiated cell into apatient.

Differentiated Cells

Differentiated cells, such as terminally differentiated cells, may bederived from the progenitor cells or cell lines made according to themethods described. We therefore disclose methods for generatingdifferentiated cells, the methods comprising generating progenitor cellsor cell lines as described, and deriving differentiated cells fromthese.

Differentiated cells which may be made according to the methodsdescribed here may include any or all of the following:

i) adipocyte: the functional cell type of fat, or adipose tissue, thatis found throughout the body, particularly under the skin. Adipocytesstore and synthesize fat for energy, thermal regulation and cushioningagainst mechanical shock

ii) cardiomyocytes: the functional muscle cell type of the heart thatallows it to beat continuously and rhythmically

iii) chondrocyte: the functional cell type that makes cartilage forjoints, ear canals, trachea, epiglottis, larynx, the discs betweenvertebrae and the ends of ribs

iv) fibroblast: a connective or support cell found within most tissuesof the body. Fibroblasts provide an instructive support scaffold to helpthe functional cell types of a specific organ perform correctly.

v) hepatocyte: the functional cell type of the liver that makes enzymesfor detoxifying metabolic waste, destroying red blood cells andreclaiming their constituents, and the synthesis of proteins for theblood plasma

vi) hematopoietic cell: the functional cell type that makes blood.Hematopoietic cells are found within the bone marrow of adults. In thefetus, hematopoietic cells are found within the liver, spleen, bonemarrow and support tissues surrounding the fetus in the womb.

vii) myocyte: the functional cell type of muscles

viii) neuron: the functional cell type of the brain that is specializedin conducting impulses

ix) osteoblast: the functional cell type responsible for making bone

x) islet cell: the functional cell of the pancreas that is responsiblefor secreting insulin, glucogon, gastrin and somatostatin. Together,these molecules regulate a number of processes including carbohydrateand fat metabolism, blood glucose levels and acid secretions into thestomach.

Uses of Progenitor Cells and Differentiated Cells

Progenitor cell lines and differentiated cells made according to themethods and compositions described here may be used for a variety ofcommercially important research, diagnostic, and therapeutic purposes.

For example, populations of undifferentiated cells may be used toprepare antibodies and cDNA libraries that are specific for thedifferentiated phenotype. General techniques used in raising, purifyingand modifying antibodies, and their use in immunoassays andimmunoisolation methods are described in Handbook of ExperimentalImmunology (Weir & Blackwell, eds.); Current Protocols in Immunology(Coligan et al., eds.); and Methods of Immunological Analysis (Masseyeffet al., eds., Weinheim: VCH Verlags GmbH). General techniques involvedin preparation of mRNA and cDNA libraries are described in RNAMethodologies: A Laboratory Guide for Isolation and Characterization (R.E. Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell &Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey,eds., 2000). Relatively homogeneous cell populations are particularlysuited for use in drug screening and therapeutic applications.

These and other uses of progenitor cell lines and differentiated cellsare described in further detail below, and elsewhere in this document.The progenitor cell lines and differentiated cells may in particular beused for the preparation of a pharmaceutical composition for thetreatment of disease. Such disease may comprise a disease treatable byregenerative therapy, including cardiac failure, bone marrow disease,skin disease, burns, degenerative disease such as diabetes, Alzheimer'sdisease, Parkinson's disease, etc and cancer.

Drug Screening

Progenitor cell lines and differentiated cells made according to themethods and compositions described here may also be used to screen forfactors (such as solvents, small molecule drugs, peptides,polynucleotides, and the like) or environmental conditions (such asculture conditions or manipulation) that affect the characteristics ofdifferentiated cells.

In some applications, progenitor cell lines and differentiated cells areused to screen factors that promote maturation, or promote proliferationand maintenance of such cells in long-term culture. For example,candidate maturation factors or growth factors are tested by adding themto progenitor cells or differentiated cells in different wells, and thendetermining any phenotypic change that results, according to desirablecriteria for further culture and use of the cells.

Furthermore, gene expression profiling of progenitor cell lines anddifferentiated cells may be used to identify receptors, transcriptionfactors, and signaling molecules that are unique or highly expressed inthese cells. Specific ligands, small molecule inhibitors or activatorsfor the receptors, transcription factors and signaling molecules may beused to modulate differentiation and properties of progenitor cell linesand differentiated cells.

Particular screening applications relate to the testing ofpharmaceutical compounds in drug research. The reader is referredgenerally to the standard textbook “In vitro Methods in PharmaceuticalResearch”, Academic Press, 1997, and U.S. Pat. No. 5,030,015), as wellas the general description of drug screens elsewhere in this document.Assessment of the activity of candidate pharmaceutical compoundsgenerally involves combining the differentiated cells with the candidatecompound, determining any change in the morphology, marker phenotype, ormetabolic activity of the cells that is attributable to the compound(compared with untreated cells or cells treated with an inert compound),and then correlating the effect of the compound with the observedchange.

The screening may be done, for example, either because the compound isdesigned to have a pharmacological effect on certain cell types, orbecause a compound designed to have effects elsewhere may haveunintended side effects. Two or more drugs can be tested in combination(by combining with the cells either simultaneously or sequentially), todetect possible drug—drug interaction effects. In some applications,compounds are screened initially for potential toxicity (Castell et al.,pp. 375-410 in “In vitro Methods in Pharmaceutical Research,” AcademicPress, 1997). Cytotoxicity can be determined in the first instance bythe effect on cell viability, survival, morphology, and expression orrelease of certain markers, receptors or enzymes. Effects of a drug onchromosomal DNA can be determined by measuring DNA synthesis or repair.[³H]thymidine or BrdU incorporation, especially at unscheduled times inthe cell cycle, or above the level required for cell replication, isconsistent with a drug effect. Unwanted effects can also include unusualrates of sister chromatid exchange, determined by metaphase spread. Thereader is referred to A. Vickers (PP 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

Tissue Regeneration

Progenitor cell lines and differentiated cells made according to themethods and compositions described here may also be used for tissuereconstitution or regeneration in a human patient in need thereof. Thecells are administered in a manner that permits them to graft to theintended tissue site and reconstitute or regenerate the functionallydeficient area.

For example, the methods and compositions described here may be used tomodulate the differentiation of stem cells. Progenitor cell lines anddifferentiated cells may be used for tissue engineering, such as for thegrowing of skin grafts. Modulation of stem cell differentiation may beused for the bioengineering of artificial organs or tissues, or forprosthetics, such as stents.

In another example, neural progenitor cells are transplanted directlyinto parenchymal or intrathecal sites of the central nervous system,according to the disease being treated. Grafts are done using singlecell suspension or small aggregates at a density of 25,000-500,000 cellsper μL (U.S. Pat. No. 5,968,829). The efficacy of neural celltransplants can be assessed in a rat model for acutely injured spinalcord as described by McDonald et al. (Nat. Med. 5:1410, 1999. Asuccessful transplant will show transplant-derived cells present in thelesion 2-5 weeks later, differentiated into astrocytes,oligodendrocytes, and/or neurons, and migrating along the cord from thelesioned end, and an improvement in gate, coordination, andweight-bearing.

Certain neural progenitor cells are designed for treatment of acute orchronic damage to the nervous system. For example, excitotoxicity hasbeen implicated in a variety of conditions including epilepsy, stroke,ischemia, Huntington's disease, Parkinson's disease and Alzheimer'sdisease. Certain differentiated cells as made according to the methodsdescribed here may also be appropriate for treating dysmyelinatingdisorders, such as Pelizaeus-Merzbacher disease, multiple sclerosis,leukodystrophies, neuritis and neuropathies. Appropriate for thesepurposes are cell cultures enriched in oligodendrocytes oroligodendrocyte precursors to promote remyelination.

Hepatocytes and hepatocyte precursors prepared using our methods can beassessed in animal models for ability to repair liver damage. One suchexample is damage caused by intraperitoneal injection of D-galactosamine(Dabeva et al., Am. J. Pathol. 143:1606, 1993). Efficacy of treatmentcan be determined by immunohistochemical staining for liver cellmarkers, microscopic determination of whether canalicular structuresform in growing tissue, and the ability of the treatment to restoresynthesis of liver-specific proteins. Liver cells can be used in therapyby direct administration, or as part of a bioassist device that providestemporary liver function while the subject's liver tissue regeneratesitself following fulminant hepatic failure.

The efficacy of cardiomyocytes prepared according to the methodsdescribed here can be assessed in animal models for cardiac cryoinjury,which causes 55% of the left ventricular wall tissue to become scartissue without treatment (Li et al., Ann. Thorac. Surg. 62:654, 1996;Sakai et al., Ann. Thorac. Surg. 8:2074, 1999, Sakai et al., J. Thorac.Cardiovasc. Surg. 118:715, 1999). Successful treatment will reduce thearea of the scar, limit scar expansion, and improve heart function asdetermined by systolic, diastolic, and developed pressure. Cardiacinjury can also be modeled using an embolization coil in the distalportion of the left anterior descending artery (Watanabe et al., CellTransplant. 7:239, 1998), and efficacy of treatment can be evaluated byhistology and cardiac function. Cardiomyocyte preparations can be usedin therapy to regenerate cardiac muscle and treat insufficient cardiacfunction (U.S. Pat. No. 5,919,449 and WO 99/03973).

Cancer

Progenitor cell lines and differentiated cells made by the methods andcompositions described here may be used for the treatment of cancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

More particular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastric cancer,pancreatic cancer, glial cell tumors such as glioblastoma andneurofibromatosis, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer,renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer. Further examplesare solid tumor cancer including colon cancer, breast cancer, lungcancer and prostrate cancer, hematopoietic malignancies includingleukemias and lymphomas, Hodgkin's disease, aplastic anemia, skin cancerand familiar adenomatous polyposis. Further examples include brainneoplasms, colorectal neoplasms, breast neoplasms, cervix neoplasms, eyeneoplasms, liver neoplasms, lung neoplasms, pancreatic neoplasms,ovarian neoplasms, prostatic neoplasms, skin neoplasms, testicularneoplasms, neoplasms, bone neoplasms, trophoblastic neoplasms, fallopiantube neoplasms, rectal neoplasms, colonic neoplasms, kidney neoplasms,stomach neoplasms, and parathyroid neoplasms. Breast cancer, prostatecancer, pancreatic cancer, colorectal cancer, lung cancer, malignantmelanoma, leukaemia, lympyhoma, ovarian cancer, cervical cancer andbiliary tract carcinoma are also included.

In preferred embodiments, the progenitor cell lines and differentiatedcells made according to the methods and compositions described here areused to treat T cell lymphoma, melanoma or lung cancer.

The progenitor cell lines and differentiated cells made according to themethods and compositions described here may also be used in combinationwith anticancer agents such as endostatin and angiostatin or cytotoxicagents or chemotherapeutic agent. For example, drugs such as such asadriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere andalkaloids, such as vincristine, and antimetabolites such asmethotrexate. The term “cytotoxic agent” as used herein refers to asubstance that inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g. I, Y, Pr), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

Also, the term includes oncogene product/tyrosine kinase inhibitors,such as the bicyclic ansamycins disclosed in WO 94/22867;1,2-bis(arylamino)benzoic acid derivatives disclosed in EP 600832;6,7-diamino-phthalazin-1-one derivatives disclosed in EP 600831;4,5-bis(arylamino)-phthalimide derivatives as disclosed in EP 516598; orpeptides which inhibit binding of a tyrosine kinase to a SH2-containingsubstrate protein (see WO 94/07913, for example). A “chemotherapeuticagent” is a chemical compound useful in the treatment of cancer.Examples of chemotherapeutic agents include Adriamycin, Doxorubicin,5-Fluorouracil (5-FU), Cytosine arabinoside (Ara-C), Cyclophosphamide,Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan,Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,Mitoxantrone, Vincristine, VP-16, Vinorelbine, Carboplatin, Teniposide,Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,Nicotinamide, Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan andother related nitrogen mustards, and endocrine therapies (such asdiethylstilbestrol (DES), Tamoxifen, LHRH antagonizing drugs,progestins, anti-progestins etc).

Stem Cells

As used in this document, the term “stem cell” refers to a cell that ondivision faces two developmental options: the daughter cells can beidentical to the original cell (self-renewal) or they may be theprogenitors of more specialised cell types (differentiation). The stemcell is therefore capable of adopting one or other pathway (a furtherpathway exists in which one of each cell type can be formed). Stem cellsare therefore cells which are not terminally differentiated and are ableto produce cells of other types.

Stem cells as referred to in this document may include totipotent stemcells, pluripotent stem cells, and multipotent stem cells.

Totipotent Stem Cells

The term “totipotent” cell refers to a cell which has the potential tobecome any cell type in the adult body, or any cell of theextraembryonic membranes (e.g., placenta). Thus, the only totipotentcells are the fertilized egg and the first 4 or so cells produced by itscleavage.

Pluripotent Stem Cells

“Pluripotent stem cells” are true stem cells, with the potential to makeany differentiated cell in the body. However, they cannot contribute tomaking the extraembryonic membranes which are derived from thetrophoblast. Several types of pluripotent stem cells have been found.

Embryonic Stem Cells

Embryonic Stem (ES) cells may be isolated from the inner cell mass (ICM)of the blastocyst, which is the stage of embryonic development whenimplantation occurs.

Embryonic Germ Cells

Embryonic Germ (EG) cells may be isolated from the precursor to thegonads in aborted fetuses.

Embryonic Carcinoma Cells

Embryonic Carcinoma (EC) cells may be isolated from teratocarcinomas, atumor that occasionally occurs in a gonad of a fetus. Unlike the firsttwo, they are usually aneuploid. All three of these types of pluripotentstem cells can only be isolated from embryonic or fetal tissue and canbe grown in culture. Methods are known in the art which prevent thesepluripotent cells from differentiating.

Adult Stem Cells

Adult stem cells comprise a wide variety of types including neuronal,skin and the blood forming stem cells which are the active component inbone marrow transplantation. These latter stem cell types are also theprincipal feature of umbilical cord-derived stem cells. Adult stem cellscan mature both in the laboratory and in the body into functional, morespecialised cell types although the exact number of cell types islimited by the type of stem cell chosen.

Multipotent Stem Cells

Multipotent stem cells are true stem cells but can only differentiateinto a limited number of types. For example, the bone marrow containsmultipotent stem cells that give rise to all the cells of the blood butnot to other types of cells. Multipotent stem cells are found in adultanimals. It is thought that every organ in the body (brain, liver)contains them where they can replace dead or damaged cells.

Methods of characterising stem cells are known in the art, and includethe use of standard assay methods such as clonal assay, flow cytometry,long-term culture and molecular biological techniques e.g. PCR, RT-PCRand Southern blotting.

In addition to morphological differences, human and murine pluripotentstem cells differ in their expression of a number of cell surfaceantigens (stem cell markers). Antibodies for the identification of stemcell markers including the Stage-Specific Embryonic Antigens 1 and 4(SSEA-1 and SSEA-4) and Tumor Rejection Antigen 1-60 and 1-81 (TRA-1-60,TRA-1-81) may be obtained commercially, for example from ChemiconInternational, Inc (Temecula, Calif., USA). The immunological detectionof these antigens using monoclonal antibodies has been widely used tocharacterize pluripotent stem cells (Shamblott M. J. et. al. (1998) PNAS95: 13726-13731; Schuldiner M. et. al. (2000). PNAS 97: 11307-11312;Thomson J. A. et. al. (1998). Science 282: 1145-1147; Reubinoff B. E.et. al. (2000). Nature Biotechnology 18: 399-404; Henderson J. K. et.al. (2002). Stem Cells 20: 329-337; Pera M. et. al. (2000). J. CellScience 113: 5-10.).

Sources of Stem Cells

Stem cells of various types, which may include the followingnon-limiting examples, may be used in the methods and compositionsdescribed here for producing progenitor cells, progenitor cell lines anddifferentiated cells.

U.S. Pat. No. 5,851,832 reports multipotent neural stem cells obtainedfrom brain tissue. U.S. Pat. No. 5,766,948 reports producing neuroblastsfrom newborn cerebral hemispheres. U.S. Pat. Nos. 5,654,183 and5,849,553 report the use of mammalian neural crest stem cells. U.S. Pat.No. 6,040,180 reports in vitro generation of differentiated neurons fromcultures of mammalian multipotential CNS stem cells. WO 98/50526 and WO99/01159 report generation and isolation of neuroepithelial stem cells,oligodendrocyte-astrocyte precursors, and lineage-restricted neuronalprecursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtainedfrom embryonic forebrain and cultured with a medium comprising glucose,transferrin, insulin, selenium, progesterone, and several other growthfactors.

Primary liver cell cultures can be obtained from human biopsy orsurgically excised tissue by perfusion with an appropriate combinationof collagenase and hyaluronidase. Alternatively, EP 0 953 633 A1 reportsisolating liver cells by preparing minced human liver tissue,resuspending concentrated tissue cells in a growth medium and expandingthe cells in culture. The growth medium comprises glucose, insulin,transferrin, T₃, FCS, and various tissue extracts that allow thehepatocytes to grow without malignant transformation. The cells in theliver are thought to contain specialized cells including liverparenchymal cells, Kupffer cells, sinusoidal endothelium, and bile ductepithelium, and also precursor cells (referred to as “hepatoblasts” or“oval cells”) that have the capacity to differentiate into both maturehepatocytes or biliary epithelial cells (L. E. Rogler, Am. J. Pathol.150:591, 1997; M. Alison, Current Opin. Cell Biol. 10:710, 1998; Lazaroet al., Cancer Res. 58:514, 1998).

U.S. Pat. No. 5,192,553 reports methods for isolating human neonatal orfetal hematopoietic stem or progenitor cells. U.S. Pat. No. 5,716,827reports human hematopoietic cells that are Thy-1 positive progenitors,and appropriate growth media to regenerate them in vitro. U.S. Pat. No.5,635,387 reports a method and device for culturing human hematopoieticcells and their precursors. U.S. Pat. No. 6,015,554 describes a methodof reconstituting human lymphoid and dendritic cells.

U.S. Pat. No. 5,486,359 reports homogeneous populations of humanmesenchymal stem cells that can differentiate into cells of more thanone connective tissue type, such as bone, cartilage, tendon, ligament,and dermis. They are obtained from bone marrow or periosteum. Alsoreported are culture conditions used to expand mesenchymal stem cells.WO 99/01145 reports human mesenchymal stem cells isolated fromperipheral blood of individuals treated with growth factors such asG-CSF or GM-CSF. WO 00/53795 reports adipose-derived stem cells andlattices, substantially free of adipocytes and red cells. These cellsreportedly can be expanded and cultured to produce hormones andconditioned culture media.

Stem cells of any vertebrate species can be used. Included are stemcells from humans; as well as non-human primates, domestic animals,livestock, and other non-human mammals.

Amongst the stem cells suitable for use in this invention are primatepluripotent stem (pPS) cells derived from tissue formed after gestation,such as a blastocyst, or fetal or embryonic tissue taken any time duringgestation. Non-limiting examples are primary cultures or establishedlines of embryonic stem cells.

Media and Feeder Cells

Media for isolating and propagating pPS cells can have any of severaldifferent formulas, as long as the cells obtained have the desiredcharacteristics, and can be propagated further. Suitable sources are asfollows: Dulbecco's modified Eagles medium (DMEM), Gibco #11965-092;Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco #10829-018;200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution,Gibco 11140-050; beta-mercaptoethanol, Sigma #M7522; human recombinantbasic fibroblast growth factor (bFGF), Gibco #13256-029. Exemplaryserum-containing embryonic stem (ES) medium is made with 80% DMEM(typically KO DMEM), 20% defined fetal bovine serum (FBS) not heatinactivated, 0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1mM beta-mercaptoethanol. The medium is filtered and stored at 4 degreesC. for no longer than 2 weeks. Serum-free embryonic stem (ES) medium ismade with 80% KO DMEM, 20% serum replacement, 0.1 mM non-essential aminoacids, 1 mM L-glutamine, and 0.1 mM beta-mercaptoethanol. An effectiveserum replacement is Gibco #10828-028. The medium is filtered and storedat 4 degrees C. for no longer than 2 weeks. Just before use, human bFGFis added to a final concentration of 4 ng/mL (Bodnar et al., Geron Corp,International Patent Publication WO 99/20741).

Feeder cells (where used) are propagated in mEF medium, containing 90%DMEM (Gibco #11965-092), 10% FBS (Hyclone #30071-03), and 2 mMglutamine. mEFs are propagated in T150 flasks (Corning #430825),splitting the cells 1:2 every other day with trypsin, keeping the cellssubconfluent. To prepare the feeder cell layer, cells are irradiated ata dose to inhibit proliferation but permit synthesis of importantfactors that support human embryonic stem cells (.about.4000 rads gammairradiation). Six-well culture plates (such as Falcon #304) are coatedby incubation at 37 degrees C. with 1 mL 0.5% gelatin per wellovernight, and plated with 375,000 irradiated mEFs per well. Feeder celllayers are typically used 5 h to 4 days after plating. The medium isreplaced with fresh human embryonic stem (hES) medium just beforeseeding pPS cells.

Conditions for culturing other stem cells are known, and can beoptimized appropriately according to the cell type. Media and culturetechniques for particular cell types referred to in the previous sectionare provided in the references cited.

Embryonic Stem Cells

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844,1995). Human embryonic stem (hES) cells can be prepared from humanblastocyst cells using the techniques described by Thomson et al. (U.S.Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133ff., 1998) and Reubinoff et al, Nature Biotech. 18:399,2000.

Briefly, human blastocysts are obtained from human in vivopreimplantation embryos. Alternatively, in vitro fertilized (IVF)embryos can be used, or one cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Human embryosare cultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner etal., Feral. Steril. 69:84, 1998). Blastocysts that develop are selectedfor embryonic stem cell isolation. The zona pellucida is removed fromblastocysts by brief exposure to pronase (Sigma). The inner cell massesare isolated by immunosurgery, in which blastocysts are exposed to a1:50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes,then washed for 5 minutes three times in DMEM, and exposed to a 1:5dilution of Guinea pig complement (Gibco) for 3 minutes (see Solter etal., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washesin DMEM, lysed trophectoderm cells are removed from the intact innercell mass (ICM) by gentle pipetting, and the ICM plated on mEF feederlayers.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto chimps either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Dissociated cells are replated on mEFfeeder layers in fresh embryonic stem (ES) medium, and observed forcolony formation. Colonies demonstrating undifferentiated morphology areindividually selected by micropipette, mechanically dissociated intoclumps, and replated embryonic stem cell-like morphology ischaracterized as compact colonies with apparently high nucleus tocytoplasm ratio and prominent nucleoli. Resulting embryonic stem cellsare then routinely split every 1-2 weeks by brief trypsinization,exposure to Dulbecco's PBS (without calcium or magnesium and with 2 mMEDTA), exposure to type IV collagenase (.about.200 U/mL; Gibco) or byselection of individual colonies by micropipette. Clump sizes of about50 to 100 cells are optimal.

Embryonic Germ Cells

Human Embryonic Germ (hEG) cells can be prepared from primordial germcells present in human fetal material taken about 8-11 weeks after thelast menstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S.Pat. No. 6,090,622.

Briefly, genital ridges are rinsed with isotonic buffer, then placedinto 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and cutinto <1 mm³ chunks. The tissue is then pipetted through a 100/μL tip tofurther disaggregate the cells. It is incubated at 37 degrees C. forabout 5 min, then about 3.5 mL EG growth medium is added. EG growthmedium is DMEM, 4500 mg/L D-glucose, 2200 mg/L mM sodium bicarbonate;15% embryonic stem (ES) qualified fetal calf serum (BRL); 2 mM glutamine(BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mL human recombinantleukemia inhibitory factor (LIF, Genzyme); 1-2 ng/ml human recombinantbasic fibroblast growth factor (bFGF, Genzyme); and 10 μM forskolin (in10% DMSO). In an alternative approach, EG cells are isolated usinghyaluronidase/collagenase/DNAse. Gonadal anlagen or genital ridges withmesenteries are dissected from fetal material, the genital ridges arerinsed in PBS, then placed in 0.1 ml HCD digestion solution (0.01%hyaluronidase type V, 0.002% DNAse I, 0.1% collagenase type IV, all fromSigma prepared in EG growth medium). Tissue is minced and incubated 1 hor overnight at 37 degrees C., resuspended in 1-3 mL of EG growthmedium, and plated onto a feeder layer.

Ninety-six well tissue culture plates are prepared with a sub-confluentlayer of feeder cells cultured for 3 days in modified EG growth mediumfree of LIF, bFGF or forskolin, inactivated with 5000 rad y-irradiation.Suitable feeders are STO cells (ATCC Accession No. CRL 1503). 0.2 mL ofprimary germ cell (PGC) suspension is added to each of the wells. Thefirst passage is conducted after 7-10 days in EG growth medium,transferring each well to one well of a 24-well culture dish previouslyprepared with irradiated STO mouse fibroblasts. The cells are culturedwith daily replacement of medium until cell morphology consistent withEG cells are observed, typically after 7-30 days or 1-4 passages.

Self-Renewal and Differentiation

Self-Renewal

Stem cells which are self-renewing may be identified by various meansknown in the art, for example, morphology, immunohistochemistry,molecular biology, etc.

Such stem cells preferably display increased expression of Oct4 and/orSSEA-1. Preferably, expression of any one or more of Flk-1, Tie-2 andc-kit is decreased. Stem cells which are self-renewing preferablydisplay a shortened cell cycle compared to stem cells which are notself-renewing.

For example, in the two dimensions of a standard microscopic image,human embryonic stem cells display high nuclear/cytoplasmic ratios inthe plane of the image, prominent nucleoli, and compact colony formationwith poorly discernable cell junctions. Cell lines can be karyotypedusing a standard G-banding technique (available at many clinicaldiagnostics labs that provides routine karyotyping services, such as theCytogenetics Lab at Oakland Calif.) and compared to published humankaryotypes.

Human embryonic stem and human embryonic germ cells may also becharacterized by expressed cell markers. In general, the tissue-specificmarkers discussed in this disclosure can be detected using a suitableimmunological technique—such as flow cytometry for membrane-boundmarkers, immunohistochemistry for intracellular markers, andenzyme-linked immunoassay, for markers secreted into the medium. Theexpression of protein markers can also be detected at the mRNA level byreverse transcriptase-PCR using marker-specific primers. See U.S. Pat.No. 5,843,780 for further details.

Stage-specific embryonic antigens (SSEA) are characteristic of certainembryonic cell types. Antibodies for SSEA markers are available from theDevelopmental Studies Hybridoma Bank (Bethesda Md.). Other usefulmarkers are detectable using antibodies designated Tra-1-60 and Tra-1-81(Andrews et al., Cell Lines from Human Germ Cell Tumors, in E. J.Robertson, 1987, supra). Human embryonic stem cells are typically SSEA-1negative and SSEA-4 positive, hEG cells are typically SSEA-1 positive.Differentiation of pPS cells in vitro results in the loss of SSEA-4,Tra-1-60, and Tra-1-81 expression and increased expression of SSEA-1.pPS cells can also be characterized by the presence of alkalinephosphatase activity, which can be detected by fixing the cells with 4%paraformaldehyde, and then developing with Vector Red as a substrate, asdescribed by the manufacturer (Vector Laboratories, Burlingame Calif.).

Embryonic stem cells are also typically telomerase positive and OCT-4positive. Telomerase activity can be determined using TRAP activityassay (Kim et al., Science 266:2011, 1997), using a commerciallyavailable kit (TRAPeze® XK Telomerase Detection Kit, Cat. s7707;Intergen Co., Purchase N.Y.; or TeloTAGGG™ Telomerase PCR ELISA plus,Cat. 2,013,89; Roche Diagnostics, Indianapolis). hTERT expression canalso be evaluated at the mRNA level by RT-PCR. The LightCyclerTeIoTAGGG™ hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics)is available commercially for research purposes.

Differentiation

Differentiating cells, including progenitor cell lines anddifferentiated cells derived from these, preferably display enhanceddephosphorylation of 4E-BP1 and/or S6K1. They preferably displaydecreased expression of Oct4 and/or SSEA-1. Preferably, expression ofany one or more of Flk-1, Tie-2 and c-kit is increased. Preferably,expression of any one or more of Brachyury, AFP, nestin and nurrlexpression increased. Stem cells which are self-renewing preferablydisplay a lenghtened cell cycle compared to stem cells which are notself-renewing.

Differentiating stem cells, i.e., cells which have started to, or arecommitted to a pathway of differentiation can be characterized accordingto a number of phenotypic criteria, including in particular transcriptchanges. The criteria include but are not limited to characterization ofmorphological features, detection or quantitation of expressed cellmarkers and enzymatic activity, gene expression and determination of thefunctional properties of the cells in vivo. In general, differentiatingstem cells will have one or more features of the cell type which is thefinal product of the differentiation process, i.e., the differentiatedcell. For example, if the target cell type is a muscle cell, a stem cellwhich is in the process of differentiating to such a cell will have forexample a feature of myosin expression.

In many respects, therefore, the criteria will depend on the fate of thedifferentiating stem cell, and a general description of various celltypes is provided below.

Markers of interest for differentiated or differentiating neural cellsinclude beta-tubulin EIII or neurofilament, characteristic of neurons;glial fibrillary acidic protein (GFAP), present in astrocytes;galactocerebroside (GalC) or myelin basic protein (MBP); characteristicof oligodendrocytes; OCT-4, characteristic of undifferentiated humanembryonic stem cells; nestin, characteristic of neural precursors andother cells. A2B5 and NCAM are characteristic of glial progenitors andneural progenitors, respectively. Cells can also be tested for secretionof characteristic biologically active substances. For example,GABA-secreting neurons can be identified by production of glutamic aciddecarboxylase or GABA. Dopaminergic neurons can be identified byproduction of dopa decarboxylase, dopamine, or tyrosine hydroxylase.

Markers of interest for differentiated or differentiating liver cellsinclude alpha-fetoprotein (liver progenitors); albumin, α₁-antitrypsin,glucose-6-phosphatase, cytochrome p450 activity, transferrin,asialoglycoprotein receptor, and glycogen storage (hepatocytes); CK7,CK19, and gamma-glutamyl transferase (bile epithelium). It has beenreported that hepatocyte differentiation requires the transcriptionfactor BNF-4 alpha (Li et al., Genes Dev. 14:464, 2000). Markersindependent of HNF-4 alpha expression include alpha₁-antitrypsin,alpha-fetoprotein, apoE, glucokinase, insulin growth factors 1 and 2,IGF-1 receptor, insulin receptor, and leptin. Markers dependent on HNF-4alpha expression include albumin, apoAI, apoAII, apoB, apoCIII, apoCII,aldolase B, phenylalanine hydroxylase, L-type fatty acid bindingprotein, transferrin, retinol binding protein, and erythropoietin (EPO).

Cell types in mixed cell populations derived from pPS cells can berecognized by characteristic morphology and the markers they express.For skeletal muscle: myoD, myogenin, and myf-5. For endothelial cells:PECAM (platelet endothelial cell adhesion molecule), Flk-1, tie-i,tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Forsmooth muscle cells: specific myosin heavy chain. For cardiomyocytes:GATA-4, Nkx2.5, cardiac troponin I, alpha-myosin heavy chain, and ANF.For pancreatic cells, pdx and insulin secretion. For hematopoietic cellsand their progenitors: GATA-1, CD34, AC133, β-major globulin, andβ-major globulin like gene PH1.

Certain tissue-specific markers listed in this disclosure or known inthe art can be detected by immunological techniques—such as flowimmunocytochemistry for cell-surface markers, immunohistochemistry (forexample, of fixed cells or tissue sections) for intracellular orcell-surface markers, Western blot analysis of cellular extracts, andenzyme-linked immunoassay, for cellular extracts or products secretedinto the medium. The expression of tissue-specific gene products canalso be detected at the mRNA level by Northern blot analysis, dot-blothybridization analysis, or by reverse transcriptase initiated polymerasechain reaction (RT-PCR) using sequence-specific primers in standardamplification methods. Sequence data for the particular markers listedin this disclosure can be obtained from public databases such asGenBank.

EXAMPLES Example 1 Methods: Derivation of E-RoSH Cell Lines

Embryonic stem cells (ESCs) are induced to differentiate to formembryoid bodies (Ebs) using the methycellulose-based approach describedin Lim et al, Blood. 1997; 90:1291-1299)

Day 3 to day 6 embryoid bodies are harvested, dissociated into singlecell suspensions by collagenase digestion (Robertson E J. Embryo-derivedstem cell lines. In: Robertson E J, ed. Teratocarcinomas and embryonicstem cells: a practical approach. Oxford: IRL Press Limited;1987:71-112) and plated on at a density of 1-5×10⁵cells per 10 cm feederplate. After about a week, the cells proliferated and differentiatedinto a complex mixture of cell types.

Colonies of rapidly dividing cells resembling embryo-derived RoSH cellsare picked and expanded sequentially to a 48-well plate, 24-well plate,6-well plate and then a 10 cm plate. The culture from each colony isnamed E-RoSH1, 2, 3 . . . in the sequence in which each culture isestablished.

Each of these cell cultures are then replated at 10-100 cells per 10 cmplate. Colonies are then selected and expanded to establish sublinesthat are named based on their parental lines e.g. E-RoSH1.1, 1.2, 1.3,etc. For suspension cultures, 1×10⁶ cells are plated on 10 cm bacterialPetri dishes that are placed on an orbital shaker.

Alkaline phosphatase assay, and MTT assays are performed using assaykits from Chemicon (Temecula, Calif.) and Bioassay Systems (Hayward,Calif.). Chromosomes counting is performed as previously described(Robertson, supra).

Example 2 Methods: Derivation of HuES9.E Mesenchymal Stem Cell(MSC)-Like Cell Lines

HuES9 cells are cultured as previously described in Cowan et al, N EnglJ Med. 2004; 350:1353-1356.

To derive HuES9.E MSC-like cells, HuES9 cells are split 1:4 ontogelatinized feeder-free plates in using HuES9 culture media. Confluentcultures are trypsinized and split 1:4. Differentiation into adipocytes,and osteocytes is performed as previously described (Barberi et al, PLoSMed. 2005; 2:e161).

BM MSCs are prepared as previously described in Pittenger et al,Science. 1999; 284:143-147.

Genomic PCR for mouse- and human-specific repeat sequences are performedas previously described in Que et al, In Vitro Cell Dev Biol Anim. 2004;40:143-149.

Example 3 Methods: RT-PCR Analysis

Total RNA is prepared using standard protocols and are quantified using,respectively, the RiboGreen RNA Quantification kit and the PicoGreendsDNA Quantification kit (Molecular Probes, Eugene, Oreg.).

Quantitative RT-PCR is performed using TaqMan® primers (AppliedBiosystems, Foster City, Calif.).

Example 4 Methods: In vitro Endothelial Differentiation

Endothelial differentiation of E-RoSH cells and acetylated LDL uptake bydifferentiated E-RoSH cells are performed as previously described (Yinet al, Arterioscler Thromb Vasc Biol. 2004; 24:691-696)

In vitro differentiated E-RoSH vascular structures are fixed informalin, embedded in paraffin, sectioned at 4 μm and stained for vWFusing polyclonal, rabbit-generated antibody andEnvision+System-peroxidase (DakoCytomation, Gostrup, Denmark). Thesections are counterstained with Mayer's hematoxylin.

Example 5 Methods: In Vivo Endothelial Differentiation

1×10⁶ embryonic stem cells are transplanted subcutaneously into SCIDmice. At three weeks when embryonic stem cell-derived tumors are about 1cm in diameter, 1×10⁵ E-RoSH cells labeled with Qdot® nanocrystals (655nm emission) using a Qtracker® Cell Labeling Kit (Quantum Dot Corp,Hayward, Calif.) are injected into the embryonic stem cell-derivedteratoma.

Three days later, the mice are euthanized with an overdose of anesthesiaand the tumors are removed. The tumors are fixed in 4% paraformaldehydeand cryosectioned at 20 μm thickness. The sections are assayed forpecam-1 immunoreactivity using rat anit-pecam1 (Pharmingen, San Diego,Calif.) followed by FITC-conjugated rabbit anti-rat antibody (Chemicon,Temecula, Calif.), and counterstained with DAPI. The sections areanalyzed by confocal microscopy.

Example 6 Derivation of Lineage-Restricted Endothelial Progenitor CellLines from Mouse Embryonic Stem Cells (mESCs)

To derive endothelial progenitor cell lines from mouse embryonic stemcells (mESCs), we relied on our previous experience of deriving RoSHendothelial progenitor cell lines from 5.5 dpc delayed blastocysts andearly post-implantation mouse embryos (Yin et al, Arterioscler ThrombVasc Biol. 2004; 24:691-696).

We rationalized that since differentiation of embryonic stem cells(ESCs) into embryoid bodies (EBs) recapitulates some of early events inmammalian development, 3 to 6 days old embryoid bodies that aredevelopmentally analogous to 5.5 dpc delayed blastocysts and earlypost-implantation embryos, will be enriched for cells that gave rise toRoSH progenitor cells. Therefore,

3 to 6 day old embryoid bodies are generated using a semi-solid,methycellulose-based media (Lim et al, Blood. 1997; 90:1291-1299),dissociated into cell suspensions by collagenase digestion to disruptthe differentiating microenvironment of the embryoid bodies, and platedon gelatinized tissue culture plate at a density of 1-5×10⁵ cells per 10cm plate in embryonic stem (ES) media without LIF supplementation todiscourage propagation of mouse embryonic stem cells (FIG. 1A).

Propagation of dissociated cells is enhanced if they are plated onembryonic fibroblast feeder as previously noted for the derivation ofRoSH progenitor cells (Yin et al, Arterioscler Thromb Vasc Biol. 2004;24:691-696) but this tended to encourage growth of embryonic stem cells.After about a week, most of the cells differentiated into a heterogenouscell culture.

The cultures are then screened for RoSH-like colonies of rapidlydividing cells with large nucleus to cytoplasm ratio and ring-like cellsthat are immunoreactive for von Willebrand Factor (or vWF) (Yin et al,Arterioscler Thromb Vasc Biol. 2004; 24:691-696) (FIG. 1B).

Only colonies that maintained a steady rate of proliferation and astable morphology are selected when they reached a size of 2-300 cells,and expanded on either embryonic fibroblast feeder or gelatin-coatedplates to generate lines, E-RoSH 1, 2, . . . etc.

Each of these lines is then subcloned by plating the cells at a lowdensity of 10-100 cells per 10 cm plate, and colonies are then picked toderive sublines E-RoSH1.1, 1.2 etc. Alternatively, it is possible toenrich for self-renewing RoSH-like cells by passaging at 1:4 about twoor three times before cloning by plating at low density. The mostefficient yield of about one RoSH-like colony per 1-5×10⁵ embryoid bodycells is dependant on the age of embryoid bodies and the parentalembryonic stem lines. For example, D3 to D5 embryoid bodies derived fromthe E14 embryonic stem cell line and D6 embryoid bodies derived from theCSL3 embryonic stem cell line (Bourc'his et al, Science. 2001;294:2536-2539) are most efficient for derivation of RoSH-like lines.

On the other hand, derivation of RoSH-like lines from differentiatingembryonic stem cells grown in the absence of LIF or other developmentalstages of embryoid bodies is possible but much less efficient. We haveestablished nine independently derived lines, five from CSL3 embryonicstem cell line and four from E14 embryonic stem cell line.

Example 7 Characterisation of E-RoSH Endothelial Progenitor Cells

E-RoSH cells, as typified by E-RoSH2.1, are morphologically similar toembryo-derived RoSH cell lines (FIG. 1C), and unlike their parentalembryonic stem cell lines, do not have detectable alkaline phosphataseactivity (FIG. 1D).

Population doubling time is estimated be ˜15 hours by MTT assay (datanot shown). E-RoSH cells have been maintained in continuous culturefor >40 generations by passaging every two days at 1:4 to 1:5 split(data not shown).

The karyotype of E-RoSH 2.1 and 3.2 as monitored by chromosome number,is stable for at least 10 passages with a normal mean chromosome numberof 40 (FIG. 1E).

Gene expression in E-RoSH2.1 is monitored by quantitative RT-PCRanalysis of 15 genes and shown to be stable at different passages (FIG.2A). In addition, this gene expression profile is similar to that inother independently derived E-RoSH lines as well as the mouseembryo-derived RoSH lines (FIG. 2B).

Subcutaneous transplantation of E-RoSH cells into SCID or Rag1 −/−immunodeficient mice did not induce teratoma formation during a two tonine-months' observation period while similar transplantation of theparental embryonic stem cells will invariably generate a 2 cm teratomawithin three weeks, suggesting a loss of pluripotency in E-RoSH cells(data not shown).

This loss of pluripotency is further evidenced by reduced expression ofseveral genes associated with pluripotent cells such as BMP4, FGF5,Oct4, Sox-2 and Utf1 (FIG. 2C) (Wei D, Xu G L, Lin C S, Bollman B,Bestor T H. Dnmt3L and the establishment of maternal genomic imprints.Stem Cells. 2005; 23:166-185; Rao M. Dev Biol. 2004; 275:269-286).

In contrast, a set of genes consisting of runx-1, flk-1, PDGFRα, Tie-2,and c-kit whose expression is commonly associated with endothelialprogenitor cells (Jaffredo et al, Int J Dev Biol. 2005; 49:269-277), ishighly expressed (FIG. 2D).

Together these observations demonstrate that E-RoSH cells are embryonicstem cell-derivatives that are no longer pluripotent and have arestricted differentiation potential that is likely to includeendothelial potential.

Example 8 Differentiation of E-RoSH Cells Into Endothelial Cells InVitro and In Vivo

To confirm their endothelial potential, E-RoSH cells are plated onmatrigel.

Within two weeks, the cells formed a network of vascular-like tubulesthat covered the entire tissue culture dish (FIG. 3A). These tubules arepatent and cells lining the lumen endocytosed acetylated LDL (FIG. 3B)and are immunoreactive for vWF (FIG. 3C).

Expression of endothelial genes such as Tie-2 is also increased (FIG.3D).

When grown in suspension, E-RoSH cells, like embryonic stem cells,formed spherical bodies. However, unlike the tightly packed embryonicstem cell-derived embryoid bodies, E-RoSH-derived bodies aremorphologically distinct with a hollow center (FIG. 3E), providinganother distinguishing difference between embryonic stem cells andE-RoSH cells.

Genes whose expressions are associated with early lineage commitmentduring embryonic development, are significantly reduced during formationof E-RoSH-derived bodies in comparison to that during embryoid bodyformation. These genes include cerberus (cer-1) which is expressedduring early gastrulation (De Robertis et al, Int J Dev Biol. 2001;45:189-197), mixl1 which is important for axial mesendodermmorphogenesis and patterning (Hart et al, Development. 2002;129:3597-3608; Mohn et al, Dev Dyn. 2003; 226:446-459) and PCSK1 whichis expressed in neuroendocrine tissues (Seidah et al, Mol Endocrinol.1991; 5:111-122; Benjannet et al, Proc Natl Acad Sci USA. 1991;88:3564-3568) (FIG. 3F).

The relatively low expression levels of these genes are consistent withthe reduced potency of E-RoSH, and suggest that E-RoSH cells no longerhave the capacity of embryonic stem cells to differentiate into a widerepertoire of cell types from all three germ layers.

In contrast, a ten-fold increase in the expression of endothelial genessuch as c-kit and Tie-2 (FIG. 3F), suggests that E-RoSH cellspreferentially differentiate into endothelial cells with a ten-foldefficiency over its parental embryonic stem cells. Although the geneexpression of E-RoSH cells suggested that they have the potential todifferentiate into hematopoietic cells, we have not been able to inducerobust hematopoietic differentiation of these cells using standardhematopoietic differentiation assays.

When E-RoSH cells are labeled with Q-tracker, a long term,cell-permeable fluorescent cell label, and transplanted into a parentalembryonic stem cell-derived teratoma that will provide a suitablemicroenvironment for differentiating E-RoSH cells, E-RoSH cells arefound to be incorporated into the capillary plexus in the teratoma andare immunoreactive for pecam-1 (FIG. 30). Many of the transplanted cellsthat are not incorporated in the tumor vasculature are notimmunoreactive for pecam-1 (data not shown).

Example 9 Derivation of Lineage-Restricted Progenitor Cell Lines fromHuman Embryonic Stem (hES) Cell Lines

To illustrate the general applicability of our approach, we derivedMSC-like lines from huES9 a human embryonic stem cells line (Cowan etal, N Engl J Med. 2004; 350:1353-1356).

We observed that in most human embryonic stem cell cultures, humanembryonic stem cell colonies grow in a state of equilibrium withproliferating stromal fibroblastic cells (FIG. 4A) suggesting that theyare human embryonic stem cell-derived progenitor cells. To encourage thepropagation of these cells and discourage that of human embryonic stemcells, huES9 cells are cultured and passaged in the absence of feeder. Ahomogenous culture of fibroblast-like cells that are morphologicallysimilar to bone marrow derived MSC (BM-MSC) cultures is generated (FIG.4B).

Two polyclonal lines, named huES9.E1 and huES9.E3, are independentlygenerated. Unlike its parental huES9 cells, huES9.E1 did not havedetectable alkaline phosphatase activity (FIG. 4C) or express OCT4 (FIG.4D).

As our previous experience suggests that fusion between putative stemcell and feeder cell occurs to generate self-renewing cells (Que et al,In Vitro Cell Dev Biol Anim. 2004; 40:143-149), these cultures aretested and shown to be negative for mouse-specific c-mos repeatsequences but positive for human specific alu repeat sequences (FIG.4E).

The cells have 46, XX, chromosomes like its parental huES9 line (Cowanet al, N Engl J Med. 2004; 350:1353-1356) (FIG. 4F).

When grown in media supplemented with serum replacement media, thepopulation doubling time is 4-5 days, and in media supplemented with 10%fetal calf serum, the population doubling is about 36 hours.

Based on the close resemblance of huES9.E1 and huES9.E3 to BM-MSC, wefurther approximated the lineage potential of huES9.E1 by comparing itssurface antigen profile to that of BM-MSCs. These cells exhibitedtypical MSC surface markers, CD29+, CD44+, CD105+ and CD166+ (Bany andMurphy, Int J Biochem Cell Biol. 2004; 36:568-584) and did not expressCD34 and CD45 (FIG. 4F).

HuES9.E1 cells can be induced to differentiate into adipocytes andosteocytes using standard differentiation conditions (Barberi T et al,PLoS Med. 2005; 2:e161), Adipocytic differentiation is confirmed by thepresence of oil droplets in the differentiated cells and expression ofPPARγ mRNA (FIG. 4G) while osteogenesis is determined by von Kossastaining for calcium deposits in the matrix and expression ofbone-specific alkaline phosphatase, Alp1 (FIG. 4H).

Example 9 Discussion

In summary, our study provides proof that lineage-restricted embryonicstem cell-derived progenitor cell lines can be established using theprinciple that progenitor cells with their unique ability to self-renew,can be propagated without transformation. They can be distinguished fromterminally differentiating cells by their steady rate of proliferationwithout senescence.

Based on this distinguishing feature, these progenitor cells can beisolated by plating the differentiating cultures at low density toselect for steadily proliferating colonies or by continual passaging ofthe culture to select for proliferating cells while terminallydifferentiating cells will senesce and will be lost from the cultures.One requirement is that the culture media does not promote propagationof the parental embryonic stem cells.

REFERENCES

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Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments and that manymodifications and additions thereto may be made within the scope of theinvention. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the claims. Furthermore, various combinations of the features of thefollowing dependent claims can be made with the features of theindependent claims without departing from the scope of the presentinvention.

We claim:
 1. A method of deriving a human mesenchymal progenitor cellline from a parental human embryonic stem cell, wherein the mesenchymalprogenitor cell line comprises mesenchymal stem cells, the methodcomprising: (a) providing the parental embryonic stem cell ordescendents of the parental embryonic stem cell obtained by dispersingan embryonic stem cell colony with trypsin, wherein the parentalembryonic stem cell is a human embryonic stem cell; and (b) culturingthe parental embryonic stem cell in the absence of feeder cells in richmedia comprising (i) essential nutrients required for cell growth and(ii) serum or serum replacement, wherein the rich media does notcomprise additional grown regulators of hormones that promote growth ofembryonic stem cells, and wherein the culturing produces mesenchymalprogenitor cells which self-renew; thereby establishing in the absenceof transformation a mesenchymal progenitor cell line from themesenchymal progenitor cells which self-renew; wherein the mesenchymalprogenitor cell line is maintainable in cell culture for more than 20generations, and wherein the mesenchymal progenitor cell line is lineagerestricted compared to the parental embryonic stem cell.
 2. The methodaccording to claim 1, further comprising the step of selecting againstsomatic cells based on their inability to self-renew.
 3. The methodaccording to claim 1, further comprising the step of expanding themesenchymal progenitor cell line.
 4. The method according to claim 1,further comprising expanding the mesenchymal progenitor cell line in theabsence of co-culture.
 5. The method according to claim 1, furthercomprising the step of exposing the embryonic stem cell to conditionsthat enhance differentiation to a specific lineage.
 6. The methodaccording to claim 5, wherein the step of exposing the embryonic stemcell generates an embryoid body from the parental embryonic stem cell.7. The method according to claim 5, further comprising the step ofremoving the embryonic stem cells from the conditions that enhancedifferentiation after pluripotency is lost.
 8. The method according toclaim 7, wherein the step of removing comprises disaggregating anembryoid body.
 9. The method according claim 8, wherein said embryoidbody has been grown from between about 3 days to about 6 days.
 10. Themethod according to claim 1, wherein the mesenchymal progenitor cellline displays at least one of the following characteristics: (a) hasreduced expression of at least one of OCT4 or alkaline phosphataseactivity; (b) does not substantially express at least one of OCT4 oralkaline phosphatase activity; (c) is substantially non-pluripotent; (d)is substantially non-tumorigenic; (e) has reduced expression of apluripotency marker compared to an embryonic stem cell from which themesenchymal progenitor cell line is derived, the pluripotency markerselected from the group consisting of Nanog, BMP4, FGF5, OCT4, Sox-2,and Utf1; (f) is maintainable in cell culture for more than 40generations; (g) has a substantially stable karyotype or chromosomenumber when maintained in cell culture for at least 10 generations; (h)has a substantially stable gene expression pattern from generation togeneration; or (i) does not substantially induce formation of teratomawhen transplanted to a recipient animal.
 11. The method according toclaim 1, further comprising the step of deriving a differentiated cellfrom the mesenchymal progenitor cell line.
 12. The method according toclaim 1, further comprising propagating the mesenchymal progenitor cellline for at least 5 generations.
 13. The method according to claim 11,wherein the differentiated cell is an endothelial cell or a mesenchymalcell.
 14. The method according to claim 11, wherein the differentiatedcell is an adipocyte or an osteocyte.
 15. The method according to claim13, wherein expression of mesenchymal or endothelial markers of thedifferentiated cell is up-regulated.
 16. The method according to claim11, wherein expression of stem cell or pluripotency markers of thedifferentiated cell is down-regulated.
 17. The method according to claim10, wherein the recipient animal is immune compromised.
 18. The methodaccording to claim 10, wherein the mesenchymal progenitor cell line doesnot substantially induce formation of the teratoma after about threeweeks.
 19. The method according to claim 10, wherein the mesenchymalprogenitor cell line does not substantially induce formation of teratomaafter about 2 months to about 9 months.
 20. The method according toclaim 1, wherein the mesenchymal progenitor cell line is maintainable incell culture for more than 25 generations.
 21. The method according toclaim 1, wherein the mesenchymal progenitor cell line is maintainable incell culture for more than 30 generations.
 22. The method according toclaim 1, wherein the mesenchymal progenitor cell line is maintainable incell culture for more than 35 generations.
 23. The method according toclaim 1, wherein the rich media is DMEM with D-glucose, fetal calfserum, non-essential amino acids, L-glutamine, and β-mercaptoethanol.24. The method according to claim 1, wherein the mesenchymal progenitorcells which self-renew are CD29⁺, CD44⁺, CD105⁺, CD166⁺, CD34⁻, andCD45⁻.